The design of an immunogen able to induce broadly neutralizing antibodies is a major, but so far elusive, goal of HIV vaccine research. Such antibodies exist as shown by the description of rare broadly neutralizing HIV sera and a handful of broadly neutralizing human monoclonal antibodies isolated from HIV infected individuals. A number of hypotheses have been advanced to explain the difficulties associated with eliciting broadly neutralizing antibodies. These include the poor accessibility of conserved regions on the trimeric envelope spike, the target of broadly neutralizing antibodies, and mechanistic impediments to the development of appropriate B cell responses such as tolerance induction. Here it is proposed to investigate some of the requirements for the initiation of a B cell response to one of the most promising vaccine targets on HIV, the conserved gp120 CD4 binding site epitope recognized by the very well characterized broadly neutralizing antibody b12. The strategy is to clone the germline b12 heavy and light chains from DNA from the original donor and express the b12 variable domains in a B cell surface IgM antigen receptor molecule. This molecule will then be exposed to various soluble and associated forms of gp120 that may have driven the b12 response in vivo. The ability of the antigens to activate B cells will be compared in a number of standard assays. The results will provide insights into the requirements for and hindrances to triggering a b12-like broadly neutralizing antibody response. In addition, the system will provide a valuable tool for evaluating future vaccine candidates de- signed to elicit b12-like antibodies. The recent structure of a complex of b12-gp120 has provided a strong stimulus to the design of such vaccine candidates. This project aims to understand how to best trigger a neutralizing antibody response to HIV. There is widespread agreement that an HIV vaccine will need to trigger such a response to be fully effective. [unreadable] [unreadable] [unreadable]